6-monofluoromethyl-17alpha-hydroxyprogesterones and 17-acylates thereof



United States Patent 0 3,341,560 6 MONOFLUOROMET YL 17a HYDROXYPRO-GESTERONES AND 17-ACYLATES THEREOF J Allen Campbell and John E. Pike,Kalamazoo, Mich., assignors to The Upjohn Company, Kalamazoo, Mich., acorporation of Michigan 0 Drawing. Continuation of application Ser. No.143,567, Oct. 9, 1961. This application May 22, 1963, Ser. No. 282,213

2 Claims. (Cl. 260-3974) This application is a continuation of ourcopending application S.N. 143,567, filed October 9, 1961 and nowabandoned. This invention relates to certain novel steroids,

3,341 ,560 Patented Sept. 12, 1967 wherein Ac is the acyl radical of anorganic carboxylic acid, preferably a hydrocarbon carboxylic acidcontaining from one to twelve carbon atoms, inclusive.

As used in this application, the Roman numeral following the name of acompound (or compounds) indicates the relation of the compound (orcompounds) to the reaction scheme depicted above.

The novel compounds of this invention, 6u-hydroxymethyl-3 3,17 dihydroxy5a pregnan-20-one 3,17-diacylates (II),6a-fluoromethyl-3p,17a-dihydroxy-5a-pregnan-ZO-one 3,17-diacylates (IV),fi-methylene-SflJh-dihydroxy-5a-pregnan-20-one 3,17-diacylate (IVA),6afluoromethyl-3fi,17rx-dihydroxy 5a pregnan-20-one-17- acylate (V),6-methylene-3,8,17a-dihydroxy-5mpregnan- 20-one 17-acyl-ate (VA),oat-fiuoromethyl-Hot-hydroxy- 5apregnane-3,20-dione 17-acylates (VI),I-dChYdIO-fiafluoro methyl-17a-hydroxy-5a-pregnane-3,20-dione 17-acylates (XI), 6a-fluoromethyl-l7a-hydroxy-4-pregnene- 3,20-di-ne17-acylates (VIII),6a-fiuoromethyl-17a-hydroxy-1,4-pregnadiene-3,20dione 17-acylates (VII),6- fluoromethyl-17ot-hydroxy-4,6-pregnadiene-3,20-dione l7- acylates(IX), 6-fluoromethyl-17a-hydroxy-1,4,6-pregnatriene-3,20-dionel7-acylates (X) and the unacylated l7u-hydroxy compounds correspondingotherwise thereto, are highly potent progestational agents, active bothorally and parenterally. The compounds represented by Formulae II and IVto XI, inclusive, are therefore useful as progestational agents in thetreatment of humans and valuable domestic animals. They areadvantageously employed in the control of such conditions as functionaluterine bleeding and dysmenorrhea; they are useful in the maintenance ofpregnancy and the regulation of fertility. The compounds embraced byFormulae II and IV to XL'inclusive, have an improved thereapeutic ratioand are useful as anti-hormonal agents, particularly as anti-estrogensand anti-gonadotropins. They are also effective in the treatment ofacne. For these purposes, the compounds of this invention can beincorporated and administered tomammals, birds, humans and animals inthe various conventional dosage forms, such as pills, tablets, capsules,syrups or elixirs for oral use, or in liquid forms such as suspensionsand solutions which are adaptable to the natural and synthetic steroidhormones for injectable products.6u-fluoromethyl-17uhydroxy-1,4-pregnadiene-3,20-dione 17-acetate (VII)has been found to be several times as active as6u-methy1-17ahydroxy-4-pregnene 3,20 dione 17-acetate (Provera) whentested for oral progestational activity by the standard McPhail assay.

The novel compounds of the present invention are prepared from the known3 3,17u-dihydroxy-5-pregnen-20- one 3,17-diacylates (I) by the followingreactions: The 5 -pregnene compounds (I) are hydroxymethylated at the6-position of the nonconjugated double bond by reacting said compoundsunder conventional oxo reaction conditions with carbon monoxide andhydrogen in the pres? ence of a suitable catalyst under pressure toproduce a 6a-hydroxymethy1-3 5,17 a-dihYdIOXY-SwPI'CgDaII 20 ne3,17-diacylate (II).

The next step of the process of this invention involves reacting a6a-hydroxymethyl-3fi,17a-dihydroxy-5a-pregnan-20-one 3,17-diacylate (II)with a halogenating agent such as triphenylphosp'hite methiodide,triphenylphosphite hydrochloride or triphenylphosphite hydrobromide toproduce a corresponding 6-halomethyl steroid compound such as a6a-iodomethyl-3p,17a-dihydroxy-5u-pregnan-3- one 3,17-diacylate (III).

The. thus produced 6u-iodomethyl-3fi,l7a-dihydroxy- Sa-pregnan-B-One3,17-diacylate (III) is reacted with silver fluoride to yield a mixtureof a 6a-fluoromethyl-35- -17u-dihydrOxy-Su-pregnan-ZO-one 3,17-diacy1ate(IV) and a 6-methylene-3 3,l7a-dihydroxy-5u-pregnan-20-one3,17-diacylate (IVA).

The next step of the process is one employing conventional reagents andconditions in which the aboveproduced mixture of a6u-fluoromethyl-3fi,l7a-dihydroxy- 5a-pregnan-20-one 3fi,17u-diacylate(IV) and a 6-methylene-3p, l7a-dihydroxy-5a-pregnan 20 one 33,l7a-Cliacylate (IVA) undergoes hydrolysis at the 3-position byreacting with a strong mineral acid in an alkanol, e.g., concentratedsulfuric acid in absolute ethanol or concentrated hydrochloric acid inmethanol, to yield a mixture of a 6a-fiuoromethyl-33,17a-dihydroxy-5a-pregnan- 20-one-l7-acylate (V) and a N-methylene313,17cc-dlhydroxy-5a-pregnan-20-one 17-acylate (VA). The com pounds ofV and VA are separated from each other by methods well known in thesteroid art, e.g., by chromatography and crystallization.

In the next step of the process, oxidation at the 3- position of thecompounds represented by Formulae V and VA results in the conversion ofthe B s-hydroxy substituent to a keto group. Oxidation of the aforesaidcompounds (V and VA) is carried out with, e.g., sodium dichromate inacetic acid or chromic acid in glacial acetic acid or acetone, or underconventional Oppenauer conditions employing an aluminum alkoxide, e.g.,aluminum isopropoxide in the presence of a ketone such as acetone orcyclohexanane, to give a mixture of the corresponding 6a-fluoromethyl17oz hydroxy 5a pregnane 3,20- dione 17-acylates (VI) and6-1'1'16tl1Yl6I16-17a-l1Yd1'0XY- 5a-pregnane-3,20-dione l7-acylates(VIA), which can be readily separated by gradient elutionchromatography.

The step of converting the6a-fluoromethyl-17a-hydroxy-5u-pregnane-3,20-dione 17-acylates (VI) to6mfluoromethyl-17u-hydroxy-1,4-pregnadiene-3,20-dione 17- acylates (VII)can be carried out by several methods, namely by reacting a compoundrepresented by Formula VI, with a reagent chosen from the groupcomprising 1) selenium dioxide; (2) dibenzoyloxy selenium oxide in themanner disclosed in German Patent 1,079,630; (3) selenium dioxide in thepresence of a metal of the second or eighth group of the periodicsystem, e.g., magnesium, zinc, cadmium, mercury of Group II or iron,cobalt, nickel of Group VIII as disclosed in U.S. Patent 2,900,398; (4)2,4-dihalogenating agents, e.g., bromine (2 equivalents) in a mediummade acid with hydrobromic acid followed by dehydrohalogenation, e.g.,with lithium chloride and dimethylformamide or with 7-0Ollidir1e asdisclosed in U.S. Patent 2,838,531 and (5) periodic acid in accordancewith the procedure set forth in German Patent 1,042,580.

60 fiuoromethyl 17oz hydroxy-1,4-pregnadiene-3,20- dione 17-acylates(VII) can also be prepared (in the manner disclosed by Vischer et al. InExperientia IX, 371 [1953]) by growing a microorganism selected from thegenus Fusarium, e.g., Fusarium solani and Fusarium caucasicum, in anutrient medium containing assimilable non-steroidal carbon, nitrogenand phosphorus, and a compound represented by Formula VI.

6u-fluoromethyl-17a-hydroxy-5a-pregnane-3,20 dione 17-acylates (VI) canbe converted to the corresponding1-dehydro-6u-fluoromethyll7a-hydroxy-5a-pregnane 3, 20-dione 17-acylates(XI) by reaction with a 2-halogenating agent, e.g., bromine (1equivalent), followed by dehydrohalogenation, e.g., with lithiumchloride and dimethlyformamide or with 'y-collidine.

The compounds represented by Formula VI can be converted to6u-fluoromethyl-17a-hydroxy-4-pregnene-3, 20-dione 17-acylates (VIII) by2,4-dihalogenation, e.g., with bromine (2 equivalents) in an acidicmedium, followed by dehydrohalogenating the thus obtained 2,4-dihalocompound with sodium iodide. Alternatively, 6w fiuoromethyl 17a hydroxy4 pregnene 3,20-dione 17-acylates (VIII)) can be prepared (in the mannerdisclosed by Stoudt et al. in Arch. Biochem, and Biophys. 74, 280[1958]) by growing a microorganism of the genus Nocardia in a nutrientmedium containing non-steroidal carbon, nitrogen and phosphorus, and acompound represented by Formula VI.

6:: fluoromethyl 17oz hydroxy 4 pregnene 3, 20-dione 17-acylates (VIII)can be converted to the corresponding 60: fluoromethyl 17cc hydroxy 1,4-pregnadiene-3,20-dione 17-acylates (VII) by fermentation (in the mannerset forth in U.S. Patent 2,902,410) with a microorganism of the genusSeptomyxa in a nutrient medium containing non-steroidal carbon, nitrogenand phosphorus-Following the same procedure, other microorganisms can beemployed instead of Septomyxa; the family Cornybacteriaceae inclusive ofthe genera Cornybacterium, Listeria and Erysipelothrix, especially thespecies Cornybacterium simplex (ATCC 6946) and Cornybacterium hoagii(ATCC 7005), can be used in the l-dehydrogenation of the compoundsembraced by Formula VIII. Alternatively, the compounds represented byFormula VIII can be converted to those of Formula VII by heating atrefluxing temperature in a suitable solvent with selenium dioxide, orselenium dioxide in the presence of a metal of the second or eighthgroup of the periodic system in the manner of U.S. Patent 2,900,398.

The flat-hydroxy counterpart of theoat-fluoromethyll7a-hydroxy-1,4-pregnadiene-3,20 dione 17 acylates (VII)can be hydrogenated at the 1,2-position by fermentation with amicroorganism chosen from the group including ATCC 6947(Arthrobactertumescens), NRRL- B1332, ATCC 3352 (S. olivaceous) and ATCC3313 (S. cellulosae) to yield the compounds of Formula VIII. The17-acylates of Formula VII are preferably first hydrolyzed to thecorresponding 17a-hydroxy compound (VIIa), e.g., with sodium hydroxidein methanol, before being subjected to the fermentative1,2-hydrogenation. The 17-aeyl group is restored following bioconversionby esterifying the 17a-hydroxy compound (VIIIa) (in the manner set forthin U.S. Patent 2,965,541) with the selected anhydride in the presence ofan acid catalyst, e.g., p-toluenesulfonic acid, to yield a 6a-fl1101'0-methyl 17a hydroxy 4 pregnene 3,20 dione 17- acylate (VIII).

By following the procedure of Example 1 of U.S. Patent 2,889,342, 6afluoromethyl 17a hydroxy 4- pregnene-3,20-dione (VIIIa) can be convertedto 3,170;- diacyloxy-6a-fiuoromethyl-3,S-pregnadien-ZO-ones and 3, 17oz20 triacyloxy 6a fluoromethyl 3,5,20 pregnatrienes.oa-fluoromethyl-l7a-hydroxy-4-pregnene 3,20- dione 17a acylates (VIII)can be converted to 3, 17a, diacyloxy 6oz fluoromethyl 3,5 pregnadien-20-ones and 3,l7a,20-triacyloxy-6a-fiuoromethyl-3,5,20- pregnatrienes inaccordance with the procedure of Example 2 of U.S. Patent 2,889,342.

The 6st fluoromethyl 17a hydroxy 4 pregnene- 3,20-dione l7-acylates(VIII) can be converted to their corresponding 6-dehydro analogues,i.e., Got-fluoromethyl- 17a-hydroxy-4,6-pregnadiene-3,20-dionel7-acylates (1X) by heating under reflux with chloranil for a period offrom about six to about twenty four hours.

The compounds of Formula IX can be converted to 3,170; diacyloxy 6afluoromethyl 3,5,7 pregnatriene- 20 ones and 3,l7a,20 triacyloxy 6ozfluoromethyl- 3,5,7,20'-pregnatetraenes by following the procedure ofExample 2 of U.S. Patent 2,889,342.

The 17a-hydroxy counterpart of the Gd-fiUOI'OITlCthYl- 17a hydroxy 4,6pregnadiene 3,20 dione 17- acylates (D() can be dehydrogenated at the1,2-position by fermentation with a microorganism of the genus Septomyxain a nutrient medium containing non-steroidal carbon, nitrogen andphosphorus. The 17-acyl group of the compounds of Formula IX are firsthydrolyzed to the corresponding Hot-hydroxy compound (IXa), e.g., withsodium hydroxide in methanol, before being subjected to the fermentative1,2-dehydrogenation. The 17- acyl group is restored followingbioconversion by esterifying the Hot-hydroxy compound (Xa) with theselected anhydride in the presence of an acid catalyst, e.g.,p-toluenesulfonic acid, to yield a o-fiuoromethyl-17a-hydroxy-1,4,6-pregnatriene-3,20-dione 17-acylate (X) as in U.S. Patent 2,965,541.Alternatively, the compounds represented by Formula 1X can be convertedto those of Formula X by heating at refluxing temperature in a suitablesolvent with selenium dioxide, or selenium dioxide in the presence of ametal of the second or eighth group of the periodic system (in themanner of U.S. Patent 2,900,398). 7

' The 1,4,6-pregnatriene compounds represented by Formula X can also beproduced from 6u-fluoromethyl-l7ahydroxy 1,4 pregnadiene 3,20 dione17-acylates (VII) by heating under reflux with chloranil for a period offrom about six to about twenty four hours.

The starting compounds (I) employed in the present process are obtainedin the manner disclosed in Preparation IA of U.S. Patent 2,916,486 byreacting the known 31S,17a-dihydroxy-5-pregnen-20-one(flu-hydroxypregnenelone) with an anhydride of a hydrocarbon carboxylicacid containing from one to twelve carbon atoms, inclusive, in thepresence of p-toluenesulfonic acid to produce a3/3,17a-dihydroxy-5-pregnen-20-one 3,17-diacylate (I).

All of the 17'acylates embraced by Formulae VI through XI can beconverted to the corresponding 17ahydroxy compounds by saponification.For this purpose the particular 17-acylate is allowed to react inalcoholic solution at room temperature for a period of from about one toabout 24 hours with an alkali metal hydroxide suchas sodium hydroxide,potassium hydroxide; an alkali metal carbonate or bicarbonate such assodium or potassium carbonate, sodium alkaline earth metal hydroxidesuchas barium or calcium hydroxide.

To isolate the product or sulfuric acid and diluted with water andextracted with a water-immiscible organic solvent such as methylenechloride, chloroform benezene, ether or the like, and the extracts driedand evaporated to give the crude material. The crude material ganicsolvents such as methanol, ethanol, acetone, Skellysolve B (hexanes),ethyl acetate, methylene chloride or the like to give the pure17a-hydroxy compounds represented by Formulae by the esterificationthereof, e.g., the selected acid, in the manner for esterifyingditficultly esterifiable Hot-hydroxy steroids disclosed in U.S. Patent2,805,230, or in Huang-Minlon et al., I. Am. Chem, 74, 5394 (1952),i.e., by reacting a steroid containing a tertiary hydroxyl group in the170L-pOSltlOIl with the selected anhydride in the presence of an acidcatalyst, e.g., ptolu-.

enesulfonic acid.

All of the compounds embraced by Formulae I through XI can be isolatedfrom their respective reaction mixtures by conventional means, forexample, when a water-miscible solvent water and separating theresultant precipitate by filtration. Additional purification of plished.by conventional methods, for example, by elution chromatography from anadsorbent column with a mixture of suitable solvents, such as, acetone,methanol, dilute methanol, ethanol, ethylene chloride; also by gradi-jent elution chromatography from an adsorbent column with a suitablemixture of solvents, such as, methylene chloride-Skellysolve B,acetone-Skellysolve B, and the like.

EXAMPLE 1 6 m-hydroxymethyl-i 5,1 7a-dihydroxy-5a-pregnan-20 0ne3,17-diacetate (II) A mixture of g. of the known compound, 313,170!

bicarbonate or the like; an

from the reaction mixture, the latter is first neutralized with aqueousacetic, hydrochloric can then be recrystallized from orcounterparts ofthe VI through XI. The 17-acylates of the thus obtained compounds areprepared with the anhydride of is used, by pouring the reaction mixtureinto the product can be accomwas placed in a 1 gallon stainless steelautoclave equipped with a mechanical stirrer. The vessel was flushedthree times with carbon monoxide and the pressure therein brought to 450p.s.i. with carbon monoxide, then to 1150 p.s.i. with hydrogen andheated at 180 C. for a period of 18 hours. The reaction mixture wascooled and filtered through a bed of Celite (diatomaceous earth) and theclear yellow filtrate concentrated to dryness on a rotary separator. Theresidue Was triturated with a mixture of ethyl acetate and ether and thecrystals collected and washed with ether to yield 71.2 g. of productwith a melting point of 225 to 231 C. An additional 4.6 g. was obtainedfrom the mother liquor. A portion of the product was recrystallized fromethyl acetate to yield an analytical sample of6a-hydroxy-methy1-3B,17ot-dihydroxy-5apregnan-ZO-one 3,17-diacetate (II)having a melting point of 232 to 234 C. and a rotation [th, of 1(chloroform).

Analysis.Calcd. for C H O (448.58): C, 69.01; H, 8.99. Found: C, 69.64;H, 8.72.

EXAMPLE 2 6 ot-iodomethy [-3 6,1 7 a-dihydr0-xy-5 opregnan-3-0ne 3,1 7

dz'acetate (III) A suspension of 15 g. of6a-hydroxymethy1-3p,17a-dihydroxy-a-pregnan-20one 3,17-diacetate (II)and 20 g. of triphenyl-phosphite methiodide in 30 ml. of methyl iodidewas heated at refluxing temperature for a period of about 2 hours orabout one hour longer than the formation of a clear solution. Most ofthe solvent was evaporated leaving a dark syrup. The syrup Was dilutedwith a mixture of methylene chloride and ether and washed successivelywith water, dilute sodium thiosulfate, addition-al water and dried. Thesolvent was removed with a rotary evaporator to give a light-coloredoil. Addition of methanol to the oil precipitated the product which wascollected on a filter, washed With a small amount of methanol and driedto yield 12.3 g. of material melting at 202 to 204 C. An analyticalsample was obtained by recrystallization of the product from a mixtureof acetone and Skellysolve B (hexanes) to yield 6u-iodomethyl-3/3,17a-dihydroxy-5a-pregnan-3-one 3,17-diacetate (III) with a meltingpoint of 201 to 205 C. and a rotation [041 of ;+28 (chloroform).

Analysis.-Calcd. for C H O I: C, 55.91; H, 7.04; I, 22.72. Found: C,56.37; H, 7.04; I, 20.64.

EXAMPLE 3 (a) 6 afluoromethy [-36,] 7 et-dihydr0xy-5 a-pregnan-ZU- one3,17-diacetate (IV) (17) 6-methylene-3 6,17a-dIhydrOxy-Su-pregnan-ZO-one 3,17-diacetate (IVA) A solution of silverfluoride was prepared in a polyethylene bottle by condensing 26 g. ofhydrogen fluoride, adding 200 ml. of acetonitrile and then adding anexcess of silver oxide, filtering off the unreacted silver oxide andrecovering the clear silver fluoride solution. 190 ml. of the thusprepared silver fluoride solution mixed with 12 g. of 60aiodomethyl-3B,17u dihydroxy-5a-pregnan-3-one 3,17-diacetate (III) wasstirred at room temperature for a period of about one-half hour and thenheated at refluxing temperature for a period of about 2 hours. Most ofthe solvent was evaporated oil with a stream of nitrogen. The remainingconcentrated material was diluted with water and extracted thoroughlywith methylene chloride. The extracts were pooled, washed with water,dried and concentrated to dryness. The partly crystalline residue was amixture of 6a-fluoromethyl-3B,17a-dihydroxy-5apregnan-ZO-one3,17-diacetate (IV) and 6-methylene- 3B,17a-dihydroxy-5ot pregnan 20 one3,17 diacetate (IVA). The compounds of IV and IVA were separated fromeach other by conventional means, e.g., by chromatography andcrystallization.

1 0 EXAMPLE 4 (a) 6a-fluor0methyl-3fi-dihydroxy-5a-pregnan ZO-OneI7-acetate (V) 5 (b) 6-mezhylene-3BJ 7 a-dihydrOxy-S a-pregnan-ZO-one I7-acetale (VA) About 9 g. of the crude mixed products,6a-fluoromethyl-3p,17a-dihydroxy 50c pregnan-ZO-one 3,17 diacetate (IV)and 6-methylene-3fi,17ot-dihydroxy-Su-pregnan-ZO- one, 3,17-diacetate(IVA) (obtained in Example 3), were dissolved in 200 ml. of methanol and2 ml. of concentrated hydrochloric acid and refluxed for a period ofabout an hour under a stream of nitrogen. Part of the methanol wasevaporated with nitrogen, and water added to cause precipitation. Theprecipitate was separated by filtration, washed with water and dried toyield 8 g. of a mixture of6u-fluoromethyl-3/3-17ot-dihydroxy-5a-pregnan- -one 17-acet5ie (V) and6-methylene-3fi,17a-dihydroxy-5ot-pregnan-20-one 17-acetate (VA). Thecom- 20 pounds of V and VA were separated from each other byconventional means, e.g., by chromatography and crystallization.

EXAMPLE 5 b) 6-methylene-I 7a-hydroxy-5a-pregnane-3,20-di0ne I 7-acetate(VIA) 8 g. of the crude mixed products, 6a-fluoromethy1-3/3,17a-dihydroxy-5a-pregnan-20-one 17-acetate (V) and 6- methylene3,6,17a-dihydroxy-5a-pregnan-20-one 17-acetate (VA) (obtained in Example4), were added to a solution of 8 g. of sodium dichromate dihydrate in70 ml. of acetic acid. After a period of about 5 /2 hours, the reactionmixture was poured into water and the resulting precipitate filtered,washed with Water and sucked nearly dry. The precipitate was dissolvedin methylene chloride and the organic phase separated and dried overmagnesium sulfate. The methylene chloride solution was adsorbed on acolumn of 300 g. of Florisil (synthetic magnesium silicate) and thecolumn extracted by gradient elution chromatography employing 4 l. of 4%acetone in Skellysolve B and 4 l. or 10% acetone in Skellysolve B. Theresidues obtained from the evaporation of fractions 11 to 19 (400 ml.each) contained the 6-methylene compound (VIA). Recrystallization of thecombined residues gave 2 g. of product melting at 208 to 210 C. Ananalytical sample was obtained by recrystallization from a mixture ofacetone and Skellysolve B, provided pure 6- 50methylene-17a-hydroxy-5a-pregnane-3,20-dione 17 acetate (VIA) with amelting point of 213 to 215.5 C. and rotation [oc] of 24 (chloroform).

Analysis.Calcd. for C H O C, 74.57; H, 8.87. Found: C, 74.16; H, 8.91.

55 The 6a-fluoromethyl compound (VI) was eluted in fractions 23 to 29.These fractions were pooled and evaporated; the combined residues onrecrystallization gave 2 g. of product With a melting point of 172 to175 C. An analytical sample was obtained by recrystallization 60 from amixture of acetone and Skellysolve B, providing pure 6a fluoromethyl 17ahydroxy-5a-pregnane-3,20-

dione 17-acetate (IV) with a melting point of 175 to Analysis.-CalCd.for C H FO F, 4.67.

C, 70.90; H, 8.68; Found: C, 71.08; H, 8.88; F, 4.59.

EXAMPLE 6 6 ot-fluoromethyl-l 7 ot-hydroxy-I ,4-pregnadiene-3,20-di0ne I7 -acezate (I -dehydro 6 a fluoromelhyl-I 7a-hydr0xyprogesterone I7-acetate) VII) 11 heating at refluxing temperature under nitrogen fora. period of about 48 hours. The reaction mixture was concentrated to avolume of about 75 ml., diluted with methylene chloride and filteredthrough a bed of Celite. The clear filtrate was washed twice with eachof the following: freshly prepared ammonium sulfide solution, diluteammonia solution and water. Each aqueous phase was back extracted withmethylene chloride. The combined methylene chloride extracts were driedover magnesium sulfate, filtered and concentrated to a volume of about20 ml. The concentrate Was adsorbed on a column of 200 g. of Florisiland the column extracted by gradient elution chromatography employing 4l. volumes of solvent mixtures of acetone and Skellysolve B ranging inconcentration from 8% acetone in Skellysolve B to 18% acetone inSkellysolve B. Fractions of 400 ml. each were collected. Fractions 10and 11 gave a few milligrams of hy-product which by infrared spectralanalysis was identified as l-dehydro-6wfluoromethyl 17ozhydroxy-5a-pregnane-3,20- dione l7-acetate (XI);

A317,; 229 m (e=9,l25)

Fractions 14 to 21 contained the main product and were combined;concentration of these eluates followed by crystallization andrecrystallization from a mixture of acetone and Skellysolve B yielded0.4 g. of material melting at 250 to 255 C. Recrystallization of thismaterial from the same solvent system provided an analytical sample ofpure 6u-fluoromethyl-17ot-hydroxy-l,4-pregnadiene- 3,20-dione 17-acetate(VII) with a melting point of 252 to 255 C.,

[011 of+6 and Mfg- 243 m (e=9,125)

EXAMPLE 6A 6u-flu0r0methyl-1 7ot-hydr0xy-1,4-pregnadiene-3,20- dicme17-acetate (V11) A mixture of 1 g. of6a-fluoromethyl-l7a-hydroxy-5apregnane-3,20-dione l7-acetate (VI), 20ml. of t-butanol and 1.5 g. of dibenzoyloxy selenium oxide was heatedwith a water trap and reflux condenser for a period of about 12 hours.After cooling, the selenium compound was removed by filtration and thefiltrate evaporated under vacuum. The residue was taken up in ethylacetate, the extract washed with aqueous sodium carbonate solution,water and dried with wood charcoal. The extract was chromatographed overa 125 g. column of Florisil and eluted with fractions of Skellysolve Bcontaining increasing portions of acetone. The fractions containingproduct showing ultra-violet absorption at 243 me were combined,evaporated to dryness and thus obtained residue recrystallized twicefrom methanol to yield pure lightcolored crystalline6u-fluoromethyl-17a-hydroxy-1,4-pregnadiene-3,20-dione l7-acetate (VII).

EXAMPLE 7 6a-flu0romethyl-1 7a-hydr0xy-1 ,4- pregnadiene-S ,2 dione17-acetate (VII) A solution containing 320 mg. of bromine in 15 ml. ofglacial acetic acid was added with slow stirring to 410 mg. of6a-fluoromethyl-17a-hydroxy-5 a-pIegnane-SQO dione 17-acetate (VI),dissolved in 300 ml. of acetic acid to which was added 0.1ml. of 4 Nhydrogen bromide solution in acetic acid. The mixture was allowed tostand overnight and resulted in the precipitation of a crude productwhich was recovered by filtration, washed with water and dried. Thecrude material was recrystallized from a mixture of ethyl acetate andSkellysolve B to give pure light-colored2,4-dibromo-6a-fiuoromethyl-17u-hydroxy-a-pregnane-3,ZG-dione 17-acetate(VI). A solution of 100 mg. of the thus obtained 2,4-dibromo compound(VI') in 1 ml. of redistilled dimethylformamide and 100 mg. of, lithiumchloride was heated for a period of an antifoam (a mixture of about 2hours. The reactionmixture was then cooled, poured into cold water andthe aqueous solution extracted with methylene chloride. The methylenechloride extract was washed thoroughly with water, dried over anhydroussodium sulfate and concentrated in volume. The extract waschromatographed over a 25 g. column of Florisil and eluted withfractions of Skellysolve B containing increasing proportions of acetone.The fractions showing an ultraviolet absorption at his. 243 u werecombined, evaporated to dryness and the thus obtained residuerecrystallized twice from methanol to give pure light-coloredot-fluoromethyl-l7a-hydroxy-1,4-pregnadiene-3,20-dione 17-acetate (VII).

The following procedure sets forth an alternativemethod ofdehydrohalogenating the 2,4-dibromo compound (VI'). mg. of2,4-dibromo-6a-fiuoromethyl-l7a-hydroxy-5a-pregnane-3,20-dionel7-acetate (VI) was dissolved in 1 ml.of redistiiled 'y-collidine andthe solution refluxed for a period of about 40 minutes. The reactionsolution was then poured into sufficient cold dilute sulfuric acid tobind the base as the sulfate salt, and extracted with methylenechloride. The methylene chloride extract was chromatographed over a 25g. column of Florisil using Skellysolve B containing increasingproportions of acetone for elution of the column. The frac tionsexhibiting ultraviolet absorption maxima at 243 mp. were combined andcrystallized to yield pure light colored 6a. fiuoromethyl 17mhydroxy-l,4-pregnadiene-3,20- dione 17-acetate (VII).

EXAMPLE 7A 6u-flu0r0methy l-1 7a-hydr0xy-1,4-pregnadiene-3,20- dione17-acetate (VII of about 20 hours. The reaction mixture was diluted with65 ml. of water and 3 g. of sodium sulfate added. The

t-amyl alcohol was removed under vacuum and the residue extracted withchloroform. The extract was washed with water, dried over sodiumsulfate, heated to dryness on the water bath under vacuum and the solidmaterial taken up in acetone. Recrystallization of the product fromacetone yielded pure light-colored crystalline6a-fluoromethyl-lh-hydroxy-IA pregnadiene- 3,20-dione l7- acetate (VII).

EXAMPLE 7B 6 a-fluoromethy l-l 7a-hydr0xy-1 ,4-pregnad iene3 ,2 0- dione17-acetate (VII) Six 100-milliliter portions of a medium in250-milliliter Erlenmeyer flasks containing one percent glucose, twopercent corn steep liquor (60 percent solids) and tap water was adjustedto a pH of 4.0. This medium was sterilized for 45 minutes at fifteenpounds per square inch pressure and inoculated with a one to two daysgrowth of Fusarium solani A.T.C.C.ll712. The Erlenmeyer flasks wereshaken at room temperature at about 24 degrees centigrade for a periodof three days. At the end of this period, this 600-milliliter volume wasused as an inoculum for ten liters of the same glucose-corn steep liquormedium which in addition contained ten milliliters of lard oil andoctadecanol). The fermentor was placed into the water bath, adjusted to28 degrees centigrade, and the contents stirred (300 rpm.) and aerated(0.5 l. air/l0 1. beer). After 17 hours of incubation, when a goodgrowth developed and the alkalinity rose to pH 6.7, two grams of6u-fluoromethyl-17ot-hy droxy-a-pregnane-3,20-dione 17-acetate (VI) plusone gram of 3-ketobisnor-4-cholen-22-al, dissolved in 115 milliliters ofdimethylformamide, was added and the incubation (conversion) carried outat the same temperature and aeration for 48 hours (final pH 7.9). Themycelium was filtered off and the steroidal material was extracted withmethylene chloride, the methylene extracts evapo rated to dryness, andthe resulting residue chromatographed over a Florisil column. The columnwas packed with 200 grams of Florisil and was developed with five40-0-milliliter fractions each of methylene chloride, SkellysolveB-acetone mixture of 9:1, 8:2, 7:3, 1:1 and methanol. The fractioneluted with Skellysolve B-acetone (7.3) on recrystallization fromacetone gave the desired product, 6a-1luoromethyl-17ahydroxy-1,4-pregnadiene-3,20 dione 17-acetate (VII).

Following the procedure of Example 7B, but instead of F usarium SOIGIZZ'ATCC. 11712, employing Fusarium solani A.T.C.C. 10915 or Fusariumcaucasz'cu-m No. 5978 of the Fungi Culture Collection of the Institutefor Fermentation of Takeda Pharmaceutical Industries, Ltd., Osaka,Japan, is likewise productive of6a-fluoromethyll7a-hydroxy-1,4-pregnadiene-3,20 dione 17-acetate (VII).

EXAMPLE 8 1 -d ehydr0-6 et-fluorometh y [-1 7 a-h ydr0xy-5 tit-pregnanc-3,20-di011e 17-acetate (XI) A solution containing 160 mg. of bromine in10 ml. of glacial acetic acid was added with slow stirring to 410 mg. of6oc-fluoromethyl-l7a-hydroxy-5u-pregnane- 3,20-dione 17-acetate (VI),dissolved in 300ml. of acetic acid to which was added 0.1 ml. of 4 Nhydrogen bromide solution in acetic acid. The mixture was allowed tostand overnight and resulted in the precipitation of a crude productwhich was recovered by filtration, washed with water and dried. Thecrude material was recrystallized from a mixture of ethyl acetate andSkellysolve B to give pure light-colored2-bromo-6a-fiuoromethyl-17a-hydroxy- 5ot-pregnane-3,20-dione 17a-acetate(VI"). A solution of 100 mg. of the thus obtained 2-bromo compound (VI")in 1 ml. of redistilled dimethylformamide and 100 mg. of lithiumchloride was heated for a period of about 2 hours. The reaction mixturewas then cooled, poured into cold water and the aqueous solutionextracted with methylene chloride. The methylene chloride extract waswashed thoroughly with water, dried over anhydrous sodium sulfate andconcentrated in volume. The extract was chromatographed over a 25 g.column of Florisil and eluted with fractions of Skellysolve B containingincreasing proportions of acetone. The fractions showing an ultravioletabsorption at were combined, evaporated to dryness and the thus obtainedresidue recrystallized twice from methanol to give pure light colored1-dehydro-6u-fluoromethyl-170thydroxy-5a-pregnane-3,ZO-dione l7-acetate(XI).

The following procedure sets forth an alternative method ofdehydrohalogenating the bromo compound (VI"). 100 mg. of2-bromo-6a-fiuoromethyl-17u-hydroxy-5a-pregnane-3,20-dione 17-acetate(VI") was dissolved in -1 ml. of redistilled 'y-collidine and thesolution refluxed for a period of about 40 minutes. The reactionsolution-was then poured into sufficient cold dilute sulfuric acid tobind the base as the sulfate salt, and extracted with methylenechloride. The methylene chloride extract was chromatographed over a 25g. column of Florisil using Skellysolve B containing increasingproportions of acetone for elution of the column. The fractionsexhibiting ultraviolet absorption maxima at 229 m (6 ,125) were combinedand crystallized to yield pure light colored1-dehydro-6a-fluoromethyl-l7a-hydroxy-5upregnane-3,20-dione 17-acetate(XI).

1 4 EXAMPLE 9 6 u-fluoromethy [-1 7 a-lz ydr0xy-4 -pregnene-3 ,ZO-dione17-acetate (VIII) 410 mg. of 6a-fiuoromethyl-l7ot-hydroxy-5a-pregnane-3,20-dione 17-acetate (VI) in 10 ml. of dioxane was acidified with adrop of 4 N hydrogen bromide in dioxane and 320mg. of bromine added overa period of 1 minute. After a period of about 1 hour at roomtemperature, an excess of sodium bicarbonate solution was added to thereaction mixture. The precipitated 2,4-dibromo derivative of 60:fluoromethyl 17u-hydroxy-5a-pregnan-3,ZO-dione 17-acetate (VI) wastreated with 0.9 g. of sodium iodide in 15 ml. of acetone containingbromoacetone, and the mixture heated at refluxing temperature for aperiod of about 2.5 hours. 0.3 g. of oxalic acid was then added andheating continued for a period of about 1 hour. After cooling, ethylacetate was added and the solution filtered. The filtrate was washedwith water and sodium bicarbonate solution, then dried with sodiumsulfate. The filtrate was stirred with 500 mg. of zinc dust in 2 ml. ofacetic acid for about 1 hour and then filtered. The organic layer waswashed successively with water, sodium bicarbonate solution and driedwith sodium sulfate. Evaporation of the solvent gave the crude eo-unsaturated ketone, which on purification with the Girard reagent,followed by subsequent crystallization yielded pure light colored 6afiuoromethyl -17et-hydroxy-4-pregnene-3,20- dione 17-acetate (VIII).Alternatively, if desired, the crude c p-unsaturated ketone can bepurified by chromatography over Florisil with increasing proportions ofacetone is Skellysolve B, followed by recrystallization.

EXAMPLE 10 6 a-fluoromethyl-I 7 ot-/zydr0xy-4 -pregna ne-3 ,2 O-dione I7 -acetate VIII) A medium consisting of 1% dextrose hydrate, 2%cornsteep liquor of 60% solids and tap water was adjusted to pH 4.9 withsodium hydroxide. The medium was steam sterilized at 15 pounds pressurefor about 30 minutes, cooled and then inoculated with a 24 hour growth,from spores, of'Nocardz'a blackwellii (NCTC 630 [Medical ResearchCouncil of the Lister Institute, London]). The medium was agitated andsparged with sterile air at the rate of one-tenth volume of air pervolume of medium per minute. At the end of 24 hours of fermentation atroom temperature, the pH was about 7.4. To this culture, there was addeda solution of 6Dt-flUOI'OII16lIhYl-17OL-IIY-droxy-Sa-pregnane-3,20-dione 17-acetate (VI) dissolved in a minimalamount of dimethylformamide. The solution was prepared by dissolving 5parts of the steroid in 100 parts of the solvent and adding about 10 ml.of the solution per liter of the medium. Fermentation was continued fora period of about 6 hours whereupon the mycelium and beer were extractedthoroughly with methylene chloride. The extract was washed with sodiumbicarbonate solution and then with water, dried and concentrated undervacuum to give 6a-fluoromethyl-17othydroxy-4-pregnene-3,ZO-dione(VIIIa).

Instead of Nocardia blackwellii (NCTC 630 [Medical Research Council ofthe Lister Institute, London]) used in Example 10 to producefermentative dehydrogenation at the 4,5-position, other microorganismsmay be similarly effectively employed; included are those chosen fromthe group consisting of: ATCC 4275 (Nocardia convo luta); ATCC 9604(Nocardia'gardneri) and NRRL B1365 (Nocardia coeliaca).

A solution composed of 1 g. of6a-fluoromethyl-l7ochydroxy-4-pregnene-3,20-dione (VIIIa), 2.5 ml. ofdistilled acetic anhydride, 250 mg. of p-toluenesulfonic acid and 2.5ml. of acetic acid was stirred for a period of about minutes. Themixture was poured with vigorous stirring into water. The precipitatedsolid was separated by filtration, dried, chromatographed over Florisilwith increasing proportions of acetone in Skellysolve B andrecrystallized from ethyl acetate to yield light-colored6a-fiuoromethyl-l7ot-hydroxy-4-pregnene-3,20-dione l7- acetate (VIII).

Substituting another lower-hydrocarbon carboxylic acid anhydride, forthe acetic anhydride is productive of other 6w fluoromethyl 17a hydroxy4 pregnene- 3,20-dione '17-acylates (VIII) wherein the acyl radical ofthe acylate group is the acyl radical of, for example, a lower-aliphaticacid, e.g., formic (formic acid plus acetic anhydride) propionic,butyric, isobutyric, caproic, valeric, isovaleric, trimethylacetic,2-methylbutyric, 3ethylbutyric, hexanoic, diethylacetic, triethylaceticheptanoic, octanoic, a-ethylisovaleric, a cyclic acid, e.g.,cyclopropylideneacetic, a cycloaliphatic acid, e.g., cyclopentylformic,cyclo pentylacetic, fi-cyclopentylpropionic, cyclohexylformic,cyclohexylacetic, p-cyclohexylpropionic, an aryl or alkaryl acid, e.g.,benzoic, methylbenzoic, dirnethylbenzoic, ethylbenzoic,trimethylbenzoic, cit-naphthoic, 3-methyl-anaphthoic, an aralkyl acid,e.g., phenylacetic, phenylpropicnic, diphenylacetic, and triphenylaceticacid.

EXAMPLE A 6a-fluor0methyl-17a-hydr0xy-1,4-pregnadiene-3,20- dione I7-acetate (VII) Six IUD-milliliter portions of a medium in250-milliliter Erlenmeyer flasks containing one percent glucose, twopercent corn steep liquor (60 percent solids) and tap water was adjustedto a pH of 4.9. This medium was sterilized for 45 minutes at fifteenpounds per square inch pressure and inoculated with a one to two daygrowth of Septomyxa aflinis ATCC. 6737. The Erlenmeyer flasks wereshaken at room temperature at about 24 degrees centigrade for a periodof three days. At the end of this period, this 600-milliliter volume wasused as an inoculum for ten liters of the same glucose-corn steep liquormedium which in addition contained ten milliliters of an antitoarn (amixture of lard oil and octadecanol). The fermentor was placed into thewater bath, adjusted to 28 degrees centigrade, and the contents stirred(300 r.p.m.) and aerated (0.5 1. air/10 1. beer). After 17 hours ofincubation, when a good growth developed and the alkalinity rose to pH6.7, two grams of 6a-fluoromethyl-17a hydroxy 4 pregnene 3,20 dione 17-acetate (VIII) plus one gram of 3-ketobisnor-4-cholen-22- a1, dissolvedin 115 milliliters of dimethylformamide, was added and the incubation(conversion) carried out at the same temperature and aeration for 24hours (final pH 7.9). The mycelium was filtered off and the steroidalmaterial was extracted with methylene chloride, the methylene extractsevaporated to dryness, and the resulting residue chromatographedover aFlorisil column. The column was packed with 200 grams of Florisil andwas developed with five 400-milliliter fractions each of methylenechloride, Skellysolve B-acetone mixtures of 9:|1, 8:2, 7:3, 1:1, andmethanol. The fraction eluted with Skellysolve B-acetone (7:3) onrecrystallization from acetone gave the desired product,6u-fluoromethy1-I7ahydroxy-1,4-pregnadiene-3,20-dione 17-acetate (VII).

Instead of by fermentative dehydrogenation, 6a-fluoromethyl 17oz hydroxy1,4 pregnadiene 3,20 dione l7-acetate (VII) or the corresponding17-hydroxy compound can be obtained by dehydrogenation of 6d-flll0l0-methyl-17a-hydroxy-4-pregnene-3,20 dione 17a acetate (VIII) or thecorresponding 17-hydroxy compound with selenium dioxide as illustratedin Example 10B.

EXAMPLE 10B 6et-flu0r0methyl-17tx-hydr0xy-1,4-pregnadiene-3,20- dione17-acetate (VII) Following the procedure of Example 10A, but employingCornybacterium simplex (ATCC 6946) instead of Septomvm afiinis, islikewise productive of 6u-fiuoro- 1,4-pregnadiene-3,20-dione 16 methyl17oz hydroxy 1,4 pregnadiene 17-acetate (VII).

EXAMPLE 10C 6 a-fluorom ethyl-1 7u-hydr0xy-I ,4-pregnadiene-3,20- dione1 7-acet te (VII) ered from the residue by recrystallization and chroma--3,20 dione tography.

0 EXAMPLE 11 6 ot-fluoromethy [-1 7 a-hydroxy-4-pregnene-3,20- dione17-acetate (VIII) A solution of 0.2 g. offiot-flIlOI'OIIlCIhYI-170t-hydIOXy- 17-acetate (VII) in 5 m1. of 1%sodium hydroxide in methanol and 2 ml. of methylene chloride was purgedwith nitrogen. After standing overnight at room temperature, thesolution was diluted with methylene chloride and washed three times withwater. The aqueous phases were back extracted with methylene chlorideand washed three times with water. The extracts were combined, dried andconcentrated to about 2 ml. and poured onto a 15 g. chromatographiccolumn of Florisil. After washing the column with increasing amounts ofacetone in Skellysolve B, the product was eluted with 2 to 8% acetone in1:1 mixtures of Skellysolve B and methylene chloride. Recrystallizationfrom a mixture of acetone and Skellysolve 'B yielded 65 mg. of6a-fluoromethyl-17a-hydroxy4l,4-pregnadiene-3,20-

dione (VIIa).

A medium consisting of 1% dextrose hydrate, 2% cornsteep liquor of 60%solids and tap water was adjusted to pH 4.9 with sodium hydroxide. Themedium was steam sterilized at 15 pounds pressure for 30 min utes,cooled, and then inoculated with a 24-hour growth, from spores, of NRRL8-1332 (Streptomyces sp.). The medium was agitated, and sparged withsterile air at the rate of one-tenth volume of airper volume of mediumper minute. At the end of 24 hours of fermentation at room temperature,the pH was about 7.4. To this culture there was added a solution of6a-fluoromethyl-l7a hydroxy-1,4-pregnadiene-3,20-dione (VIIa) dissolvedin a minimal amount of dirnethylformarnide. The solution was prepared bydissolving five parts of the steroid in parts of the solvent. and addingabout 10 m1. of the solution per liter of the medium. Fermentation wascontinued for a period of 48 hours whereuponthe mycelium and beer wereextracted thoroughly with methylene chloride. The extract was washedwith sodium bicarbonate solution and then with water, dried andconcentrated in vacuo to give 60L-flll010l'IlEIhYl-17ct-hydIOXy-4-pregnene-3,20-dione (VIIIa).

Instead of NRRL B1332 used in Example 11 to produce fermentativehydrogenation at the 1,2-position of the steroid nucleus, othermicroorganisms the group consisting cens); ATCC (S. olivaceous) and ATCC3313.

A solution composed of 1 g. of6a-fluoromethyl-l7ahydroxy-4-pregnene-3,20-dione (VIIIa), 2.5 ml. ofdistilled acetic anhydride, 250 mg. of p-toluenesulfonic acid and 2.5ml. of acetic acid was stirred for a period of about 90 minutes. Themixture was poured with vigorous stirring may be similarly effectivelyemployed; included are those chosen from t of: ATCC 6947(Arthrobactertumes- 17 into water. The precipitated solid was separatedby filtration, dried, chromatographed over Florisil with increasingproportions of acetone in Skellysolve B and recrystallized from ethylacetate to yield light-colored 6uc-fll10f0- methyl 17cc hydroxy 4pregnene 3,20 dione 17-acetate (VIII).

Other 17-acylates are prepared in a like manner by substituting one ofthe lower-hydrocarbon carboxylic acid anhydrides listed in Example inplace of acetic anhydride.

EXAMPLE 11A 3,170: diacetoxy 60c fluoromethyl 3,5 pregnadieneone and3,1700 20 triacetoxy 60a fluoromethyl- 3,5,20-pregnatriene A suspensionof 10 g. of 6o-fiuoromethyl-Not-hydroxy- 4-pregnene-3,20-dione (VIIIa)in 150 ml. of acetic anhydride is stirred at about 40 C. with 1.5 g. ofp-toluenesulfonic acid monohydrate for a period of about 10 hours. Thenabout 200 ml. of Water is added slowly with icecooling and the mixtureagitated until the excess acetic anhydride is destroyed. The crudecrystalline precipitate is filtered, washed to neutrality with water andthen Washed with a small volume of methanol. This material consists of amixture of, principally,3,17a-diacetoxy-6afluoromethyl-3,S-pregnadien-ZO-one and a minorquantity of 3,17a,20 triacetoxy 6a fluoromethyl 3,5,20- pregnatriene.Recrystallization from a 1:1 mixture of dichloromethane and methanolyields a light colored, crystalline product,3,17a-diacetoxy-6wfluoromethyl-3,5-pregnadien-20-one.

Following the procedure of Example 11A, but su'bstituting for aceticanhydride another lower-hydrocarbon carboxylic acid anhydride, e.g.,wherein the acyl radical of the acylate group is one of those listed atthe end of Example 10, yields the corresponding 3,l7a-diacyloxy- 6afluoromethyl 3,5 pregnadien 20 one and 3,1706, 20 triacyloxy 60cfluoromethyl 3,5,20 preguatriene.

EXAMPLE 11B 3,170: diacetoxy 6oz fluoromethyl 3,5 pregnadien- 20 one and3,]7a triacetoxy 6a fluoromethyl- 3,5,20-pregnatriene A suspension of 10g. of 6u-fluoromethyl-l7u-hydroxy- 4-pregnene-3,20-dione 17-acetate(VIII) in 150 ml. of acetic anhydride is stirred with 2 g. ofp-toluenesulfonic acid monohydrate at about 40 C. for a period of about16 hours. About 200 ml. of Water is then slowly added with cooling in anice bath and the mixture agitated until the excess acetic anhydride isdestroyed. The crude crystalline precipitate is filtered, washed toneutrality with Water and then washed with a small volume of methanol.This material consists of a mixture of, principally, 3,170:- diacetoxy611 fiuoromethyl 3,5 pregnadien 20 one and a minor amount of 3,170c,20triacetoxy 60c fluoromethyl 3,5,20-pregnatriene. Recrystallization froma 1:1 mixture of dichloromethane and methanol yields a light colored,crystalline product, 3,17u diacetoxy 60cfluoromethyl-3,5-pregnadien-20-one.

Following the procedure of Example 11B, but substituting for aceticanhydride another lower-hydrocarbon carboxylic acid anhydride, e.g.,wherein the acyl radical of the acylate group is one of those listed atthe end of Example 10, yields the corresponding 3,17oz-(liacyloxy 6ozfluoromethyl 3,5 pregnadien 20 one and 3,l7oc,20 triacyloxy 60cfluoromethyl 3,5,20- pregnatriene.

EXAMPLE 12 6-flu0r0methyZ-J 7 u-hydroxy-4, 6- pregnadiene-3,20-dione17-acetate (IX) 1 g. of 6w-fluoromethy1-17a-hydroxy-4-pregnene-3,20-dione 17-acetate (VIII), 1.5 g. of recrystallized 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) and 60 ml. of tertiary amylalcohol were heated to boiling under nitrogen with a few boiling chips,and gently refluxed for a period of about 6 hours. The mixture wascooled and evaporated to dryness under reduced pressure. The solidresidue (with the exception of some chloranil, which was insoluble) wasdissolved in about ml. of ether and filtered. The chloranil on thefilter paper was washed with several portions of ether and the combinedether filtrates washed with 200 ml. portions of cold 2% sodiumhydroxide. The ether filtrates were washed with cold water until thewashings were neutral, then with saturated sodium chloride solution. Thepooled ether solutions were dried over sodium sulfate and evaporated todryness. The residue crystallized readily from cold acetone to yieldpure light-colored crystalline 6-fiuoromethyl-17a-hydroxy-4,6-pregnadiene-3,20-dione 17-acetate (IX).

The ester thus obtained can be hydrolyzed with potassium hydroxide orpotassium carbonate in methanol or ethanol at room temperature in anitrogen atmosphere to give free6-fluoromethyl-17a-hydroxy-4,6-pregnadiene-3, ZO-dione (IXa).

A solution composed of 1 g. of6-fluoromethy1-17ahydroxy-4,6-pregnadien-3,ZO-dione (IXa), 2.5 ml. ofdistilled acetic anhydride, 250 mg. of p-toluenesulfonic acid and 2.5ml. of acetic acid was stirred for a period of about 90 minutes. Themixture was poured with vigorous stirring into water. The precipitatedsolid was separated by filtration, dried, chromatographed over Florisilwith increasing proportions of acetone in Skellysolve B andrecrystallized from ethyl acetate to yield light-colored 6- fluoromethyl17oz hydroxy 4,6 pregnadiene 3,20- dione 17-acetate (IX).

Other l7-acylates are prepared in a like manner by substituting one ofthe lower-hydrocarbon carboxylic acid anhydrides listed in Example 10instead of acetic anhydride.

EXAMPLE 12A 3,]7a diacetoxy 6a fluoromethyl 3,5,7 pregnatrz'ene- 20 oneand 3,17oc,20 triacetoxy 60c fluoromethyl- 3,5,7,20-tetraene Asuspension of 10 g. of 6CZ-flUOIOH16thyl-170t-hydl'OXy-4,6-pregnadiene-3,20-dione 17-acetate (IX) in ml. of acetic anhydride isstirred with 2 g. of p-toluenesulfonic acid monohydrate at about 40 C.for about 16 hours. About 200 ml. of water is then slowly added withcooling in an ice-bath and the mixture agitated until the excess aceticanhydride is destroyed. The crude crystalline precipitate is filtered,washed to neutrality with water and then washed with a small volume ofmethanol. This material consists of a mixture of, principally,3,17a-diacetoxy- 60c fluoromethyl 3,5,7 pregnatriene 20 one and a minorquantity of 3,17a,20-triacetoxy-6a-fiuoromethyl-3,5,7,20-pregnatetraene. Recrystallization from a 1:1 mixture ofdichloromethane and methanol yields a light colored, crystallineproduct, 3,17oc-diacetoxy-6wfluorornethyl- 3,5,7-pregnatriene-20-one.

Following the procedure of Example 12A, but substituting for aceticanhydride another lower-hydrocarbon carboxylic acid anhydride, e.g.,wherein the acyl radical of the acylate group is one of those listed atthe end of Example 10, yields the corresponding 3,17wdiacyloxy-6a-fluorornethyl-3,5,7 pregnatriene-2'0-one and 3,17a,20-triacyloxy-6a-fiuoromethyl-3,5,7,20-pregnatetraene.

EXAMPLE 13 6 -fluor0m ethyl-I 7 ot-lzydroxy-l ,4,6-pregnatriene-3,20-dione .Z7-acetate '(X) the end of this period, this 600 ml. volume wasused as an inoculum for 10.1 of the same glucose corn steep liquormedium which, in addition, contained 10 ml. of an antifoam (a mixture oflard oil and octadecanol). The fermentor was placed into the water bath,adjusted to 28 C., and the contents stirred (300 rpm.) and aerated(0.5 1. air per minute/ 10 1. beer). After seventeen hours ofincubation, when a good growth developed and the acidity rose to pH 6.7,1 g. 6-flu0romethyl-flat-hydroxy- 4,6-pregnadiene-3,20-dione (lXa) plus0.5 g. of 3- ketobisnor-4-cholen-22-al, dissolved in 115 ml. ofdimethylformamide, was added and the incubation (conversion) carried outat the same temperature and aeration for 24 hours (final pH 7.9). Themycelium was filtered off and the steroidal material was extracted withethyl acetate to remove the bioconversion products. The ethyl acetateextract was washed with water, dried over sodium sulfate and evaporatedto dryness. The residue was taken up in about 100 ml. of methylenechloride and chiromatographed over a column of 120 g. of Florisilsynthetic magnesium silicate. The products were eluted from the columnwith mixtures of 5 to 20% of acetone in Skellysolve B. The substanceseluted by 20% in Skellysolve B consisted of partially purified products,which was further purified by recrystallization from ace-tone to givepure light-colored crystalline 6-flu0romethyl-17'othyd-roxy-1,4,6pregnatriene-3,20 dione (Xa).

A solution composed of 1 g. of6-fluoromethyl-17uhydroxy-l,4,6-pregnatriene-3,20-dione (Xa), 2.5 ml. ofdistilled acetic anhydride, 250 mg. of p-toluenesulfonic acid and 2.5ml. of acetic acid was stirred for a period of about 90 minutes. Themixture was poured with vigorous stirring into water. The precipitatedsolid was separated by filtration, dried, chromatographed over Florisilwith increasing proportions of acetone in Skellysolve B andrecrystallized from ethyl acetate to yield light-colored 6- fluoromethylNon-hydroxy-1,4,6-pregnatriene-3,20-dione 17-acetate (X).

Other 17-acylates are prepared in alike manner by substituting one ofthe lower-hydrocarbon carboxylic acid anhydrides listed in Example 10 inplace of acetic anhydride.

EXAMPLE 14 6-flu0r0methyl-1 7a-hydr0xy-1,4,6-pregnatriene-3,20- dione17-acetate (X) A mixture of 100 milligrams of6-fluoromethyl-17ahydroxy-4,6-pregnadiene-3.20-dione 17-acetate (IX)dissolved in six milliliters of tertiary butyl alcohol and 0.55milliliter of acetic acid was heated together with thirty milligrams ofselenium dioxide to approximately 75- degrees centigrade with stirringfor a period of about 24 hours. Thereafter another thirty-milligramportion of selenium dioxide was added and the mixture heated to 75degrees centigrade under continuous stirring for a further period of 24hours. The mixture was then cooled, filtered to remove the seleniumdioxide and evaporated. The residue was chromatographed on a column ofFlorisil, eluted with mixtures of acetone and Skellysolve B andrecrystallized from acetone-Skellysolve B hexanes four times to givepure -6-fllorormethyl-l7a-hydroxy-l,4,6- prcgnatriene3,20-dione17-acetate (X), a light-colored crystalline solid.

The ester thus obtained can be hydrolyzed with potassium hydroxide orpotassium carbonate in methanol or ethanol at room temperature in anatmosphere of nitrogen to give free6-fluoromethyl-17a-1,4,6-pregnatriene-3.20-dione (Xa).

A solution composed of 1 g. of6-fluoromethyl-17ahydroxy-l,4,6,pregnatriene-3,20-dione (Xa), 2.5 ml. ofdistilled acetic anhydride, 250 mg. of p-toluenesulfonic acid and 2.5ml. of acetic acid was stirred for about 90 minutes. The mixture waspoured with vigorous stirring into water. The precipitated solid wasseparated by filtration, dried, chromatographed over Florisil withincreasing proportions of acetone in Skellysolve B and I6 crystallizedfrom ethyl acetate to yield light-colored 6 fluoromethyl1hat-hydroxy-1,4,6-pregnatriene-3,ZOdione 17-acetate (X).

Other 17-acylates are prepared in like manner by substituting one of thelower-hydrocarbon carboxylic acids listed in Example 10 instead ofacetic, anhydride.

EXAMPLE 15 1 g. of 6a-fluoromethyl-Hot-hydroxy-1,4-pregnadiene-3,20-dione 17-acetate (VII), 1.5 g. of recrystallized2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) and 60 ml. of tertiaryamyl alcohol were heated to boiling under nitrogen with a few boilingchips, and gently refluxed for a period of about 6 hours. The mixturewas cooled and evaporated to dryness under reduced pressure. The solidresidue (with the exception of some chloranil, which was insoluble) wasdissolved in about 100 ml. of ether and filtered. The chloranil on thefilter paper was Washed with several portions of ether, and the combinedether filtrates washed with 200 ml. portions of cold 2% sodiumhydroxide. The ether filtrates were washed with cold water -until thewashings were neutral, then with saturated sodium chloride solution. Thepooled ether solutions were dried over sodium sulfate and evaporated todryness. The residue crystallized readily from cold acetone to yieldpure light-colored crystalline m-fluoromethyb 17a hydroxy1,4,6-pregnatriene-3,20-dione l7-acetate (X).

The ester thus obtained can be hydrolyzed with postassiurn hydroxide orpotassium carbonate in methanol or ethanol at room temperature in anitrogen atmosphere to give free6-fiuoromethyl-l7a-hydroxy-l,4,6-pregnatriene-3,20-dione (Xa).

A solution of 1 g. of -6-fiuoromethyl-Nix-hydroxy-1,4,6-pregnatriene-3,20-dione (Xa), 2.5 ml. of distilled aceticanhydride, 250 mg. of p-toluenesultonic acid and 2.5 ml. of acetic acidwas stirred for about minutes. The mixture was poured with vigorousstirring into water.

The precipitated solid was separated by filtration, dried,

chromatographed over Florisil with increasing proportions of acetone inSkellysolve B and recrystallized from ethyl acetate to yieldlight-colored 6-fiuoromethyl- 17a hydroxy 1,4, 6-pregnatriene-3,ZO-dione17-acetate (X).

Other 17-acylates are prepared in like manner by substituting one of thelower-hydrocarbon carboxylic acids listed in Example 10 instead ofacetic anhydride.

While the compounds prepared in Examples 1 to 15 are 3,17-diacetates and17-acetates, other 3,17-diacylates and 17-acylates of these compoundscan be prepared in a similar manner by substituting as the startingmaterial a corresponding 3,l7diacylate and 17-acylate other than the3,17-diacetate and l7-acetate, wherein the acyl radical is, e.-g., thatof a lower-hydrocarbon carboxylic acid named in Example 10. i

wherein R is selected from the group consisting of hydrogen and the acylradical of a hydrocarbon car- 21 22 boxylic acid containing from one totwelve carbon atoms, 3,052,676 9/1962 Zderic et a] 260-239.55lncl'uslve- 3,087,941 4/1963 Engel 260239.55 2. 1 dehydro 60fluoromethyl-l7a-hydroxy-5a-preg- 3 178 411 4/1965 Baa1 et a1 260h 239,55 name-3,20-dione 17-acetate. n

References Cited 5 LEWIS GOTTS, Primary Examiner. UNITED STATES PATENTSELBERT L. ROBERTS, Examiner.

3,036,098 5/1962 Bowers et a1. 260397.45 J. R. BROWN, AssistantExaminer.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,341,560 September 12, 1967 J. Allan Campbell et al.

It is hereby certified that error appears in the above numbered patentrequiring correction and that the said Letters Patent should read ascorrected below.

Column 6, line 11, "N-methylene" should read 6methy' lene Column 9, line45, "C should read C column 10, line 43, "41 or 10%" should read H 4 1.of 10% line 62, "(1V)" should read (VI) Column 11, line 33, "(e=9,125)"should read (e=16,250) Column 12, line 62, "4.0" should read 4.9 Column16, line 70, "ATCC (S, olivaceous)" should read ATCC 3352 (S.olivaceous) Column 17, line 42, "3,1700" should read 3,l7 1,20-

Column 19, line 2, "10.1" should read 10 l. line 9, 1 g. 6-" should readl g. of 6- line 67, "l7 1,4,6-" should read l7a-hydroxyl,4,6

Signed and sealed this 29th day of July 1969.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. WILLIAM E SCHUYLER,JR. Attesting OfficerCommissioner of Patents

1. A COMPOUND OF THE FORMULA